method of analyzing peptide for determining c-terminal amino acid sequence

ABSTRACT

The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by applying reaction technique for successively releasing the C-terminal amino acids therefrom, which method can suppress, when releasing the C-terminal amino acids of the peptide in sequence, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide, and allows to carry out the chemical treatment thereof under widely applicable conditions. In the method according to the present invention, an alkanoic acid anhydride and a perfluoroalkanoic acid both of vapor phase, which are supplied from a mixture containing an alkanoic acid anhydride with a small amount of a perfluoroalkanoic acid added thereto, are allowed to act on a dry sample of the peptide to be examined in a dry atmosphere at a temperature chosen in a range of 15 to 60° C.; whereby the release of the C-terminal amino acid is resulted from successive formation of a 5-oxazolone structure being followed by cleavage of the 5-oxazolone ring; and then the C-terminal amino acids sequence is identified by analysis based on the decrease in molecular weight in a series of the reaction products obtained.

TECHNICAL FIELD

The present invention relates to a method for analysis of C-terminalamino acid sequence of peptide, more particularly to a method comprisingsteps of releasing the C-terminal amino acids of the peptidesuccessively by chemical means, determining the molecular weights of theobtained reaction products by mass spectrometry, and clarifying theC-terminal amino acid sequence as for the peptide, based on the observeddecreases in molecular weight that are caused by a series of amino acidseliminated successively. The present invention further relates to a kit,which is used exclusively for the above method for analysis, fortreatment to prepare the reaction products to be subjected to massspectrometry by treatment of releasing the C-terminal amino acids of thepeptide successively by chemical means.

BACKGROUND ART

With respect to peptides and proteins collected from nature, the aminoacid sequences identified thereof are essential information in course ofstudying the biological properties and functions of the peptides andproteins. Currently, the full-length amino acid sequences for peptidesand proteins are determined as deduced amino acid sequences, based oncorresponding gene information thereof, that is, nucleotide sequences ofc-DNAs produced from genomic genes or m-RNAs which encode theirpeptides. However, in identifying the c-DNAs produced from the genomicgene or m-RNA which encodes the peptide, the knowledge of partial aminoacid sequences of the peptides is still required.

It is generally considered that, as the knowledge of the partial aminoacid sequences of peptide, the N-terminal amino acid sequence andC-terminal amino acid sequence of peptide are particularly useful.Specifically explaining, for example, in selecting a c-DNA which encodesan intended peptide from a c-DNA library prepared from a large number ofm-RNAs, if the N-terminal amino acid sequence and C-terminal amino acidsequence thereof are known, the aimed c-DNA can be selected by usingnucleic acid probes that are produced based on the above amino acidsequences of the two terminals. Or, the aimed c-DNA can be amplifiedselectively by applying PCR with use of oligonucleotide primers that areproduced based on the amino acid sequences of the two termini.

As the method for analyzing the N-terminal amino acid sequence of apeptide, there has been conventionally used a method of allowing an acidto act on a peptide to release the N-terminal amino acids successivelyby hydrolysis and identifying the amino acids resulted therefrom.Meanwhile, as the method for analyzing the C-terminal amino acidsequence of a peptide, there has been proposed a method of releasing theC-terminal amino acids thereof successively by chemical means andidentifying the C-terminal amino acids released thereby, based on themolecular weight differences between the original peptide and truncatedpeptides that are obtained as reaction products therefrom. As thetechnique for releasing the C-terminal amino acids successively bychemical means, there is proposed, for example, a method comprisingsteps of allowing a vapor generated from a high concentration aqueoussolution of pentafluoropropanoic acid (CF₃CF₂COOH) or a highconcentration aqueous solution of heptafluorobutanoic acid(CF₃CF₂CF₂COOH), to act on a dried peptide under heating up condition at90° C., and releasing the C-terminal amino acids by selective hydrolysisenhanced with use of said perfluoroalkanoic acid [Tsugita, A. et al.,Eur. J. Biochem. 206, 691-696 (1992)]. There is also proposed a methodcomprising steps of using, in place of the above high concentrationaqueous solution of a perfluoroalkanoic acid, an acetonitrile solutionof pentafluoropropanoic acid anhydride [(CF₃CF₂CO)₂O] or an acetonitrilesolution of heptafluorobutanoic acid anhydride [(CF₃CF₂CF₂CO)₂O],allowing a vapor generated form the solution, to act on a dried peptideunder cooling down condition at such a low temperature, for example, at−18° C., and releasing the C-terminal amino acids selectively, which isforced with use of the perfluoroalkanoic acid anhydride [Tsugita, A. etal., Chem. Lett. 1992, 235-238; Takamoto K. et al., Eur. J. Biochem.228, 362-372 (1995)].

In said technique for selectively releasing the C-terminal amino acidsby allowing a perfluoroalkanoic acid or a perfluoroalkanoic acidanhydride, which are supplied as a vapor thereof, to act on a driedpeptide, it has been reported that an oxazolone ring structure is onceformed from the C-terminal amino acids as a reaction intermediate, in adehydration reaction shown by the following reaction scheme (I):

then, the perfluoroalkanoic acid acts on the oxazolone ring to give riseto a reaction shown by the following reaction scheme (II):

as a result, reaction of selectively releasing the C-terminal amino acidtherefrom is achieved.

As the above reaction of selectively releasing the C-terminal amino acidproceeds successively, there is obtained, at a timing when a giventreatment time has passed, a mixture comprising a series of reactionproducts in which one to ten odd amino acid residues have been removedfrom the C-terminal of the original peptide, respectively. This mixturecomprising a series of reaction products is subjected to massspectrometry to measure the masses of the ion species derived from thereaction products, whereby can be obtained a series of peaks exhibitingthe mass differences, which reflect the C-terminal amino acid sequence.Specifically explaining, the individual reaction products are formed inreaction of successively releasing C-terminal amino acids from theoriginal peptide; hence, for example, a set of reaction productsincluding several members in series, where up to several amino acidresidues have been removed from the original peptide, are subjected tomass spectrometry and, thereby, the masses of corresponding ion speciesthereto can be analyzed collectively, which enables determination ofC-terminal amino acid sequence of such several amino acid residues atone time.

Incidentally, for example, the information of C-terminal amino acidsequence used in production of nucleic acid probe or primer mayordinarily be, in terms of the nucleotide sequence which codes suchamino acid sequence, about 18 to 24 bases and accordingly about 6 to 8amino acids. The identification of C-terminal amino acid sequence of upto ten odd amino acid residues is required only in very rare cases.Therefore, the above methods for preparation of treated samplecomprising a series of reaction products, in which all the removalsextending up to 10 amino acid residues are included, by the reaction ofreleasing the C-terminal amino acids from the dried peptide, where avapor of a perfluoroalkanoic acid or a perfluoroalkanoic acid anhydrideare supplied in vapor phase and allowed to act thereon, are suitable forthe above-mentioned purposes.

DISCLOSURE OF THE INVENTION

The methods comprising such steps of supplying a vapor of aperfluoroalkanoic acid or a perfluoroalkanoic acid anhydride in vaporphase and allowing them to act on a dried peptide are useful means forclarifying the C-terminal amino acid sequence therein; however, forextending application of the methods as a general means for a wide use,the methods have been found to have the practical problems describedbelow.

In the above-mentioned method with use of a high concentration aqueoussolution of a perfluoroalkanoic acid, which allows a vapor of theperfluoroalkanoic acid to act on a dried peptide under heating upcondition, for example, at 90° C., there occurs a side reaction inwhich, at the serine residue [—NH—CH(CH₂OH)—CO—] in the peptide, anN,O-acyl rearrangement reaction proceeds between the amino group (—NH—)on its α-position and the hydroxyl group (—OH) on its β-position,subsequently, hydrolysis proceeds, which results in cleavage of peptidetaking place at the N-terminal of the serine residue. Depending upon theconditions used, there also occurs a side reaction in which, at thethreonine reside [—NH—CH(CH(CH₃)OH)—CO—] having a hydroxyl group (—OH)on its β-position, hydrolysis proceeds based on a similar mechanism,which results in cleavage of peptide taking place at the N-terminal ofthe threonine residue. There further occurs a side reaction in which, atthe aspartic acid residue [—NH—CH(CH₂COOH)—CO—] in the peptide, peptidebond rearrangement from C-terminal carboxy group to carboxy group on itsβ-position and subsequent hydrolysis proceed, which results in cleavageof peptide taking place at the C-terminal of the aspartic acid residue.

When the cleavage of peptide due to such side reactions happens,selective release of C-terminal amino acids proceeds simultaneously evento the resulting N-terminal peptide fragments. On some occasions, theco-existence of reaction products that are originated from these sidereactions may be a factor interfering with the accuracy in themeasurement by mass spectrometry conducted toward intended reactionproducts.

Further, even when there occurs no cleavage of peptide but when there isformed a branched type peptide wherein the N-terminal portion of peptideis linked to the hydroxyl group (—OH) on the β-position thereof, whichleads to loss of amide bond at the site, there is no formation ofoxazolone ring structure therefrom, and accordingly selective release ofC-terminal amino acid make no further progress thereafter.

Meanwhile, in the above-mentioned method with use of an acetonitrilesolution of a perfluoroalkanoic acid anhydride, which allows a vapor ofthe perfluoroalkanoic acid anhydride generated from the solution to acton a dried peptide under cooling down condition, for example, at −18°C., no water molecule being vaporized from the solution is present insaid system and, therefore, the method has such an advantage that theoccurrence of the above-mentioned side reactions can be avoidedeffectively. However, since the reactivity of the perfluoroalkanoic acidanhydride used is high, effective suppression of undesired sidereactions is more difficult when the treatment temperature rises higher;therefore, the treatment temperature is required to be kept at such alow temperature as, for example, −18° C. In other words, when thecontrol of the treatment temperature is not enough, there is a highpossibility that undesired side reactions are escalated thereby;therefore, in this view, the method still has somewhat weakness in thewide applicability and leaves a room to be improved further. Inaddition, when water condensation takes place in association withcooling, the resulting water gives rise to deterioration of the reagentused, i.e. deactivation of the perfluoroalkanoic acid anhydride used,which may result in a reduced reactivity on occasion, and thus thereremains some anxiety that it may happen to become a serious problem inpractical application.

The present invention solves the above-mentioned problems and aims atproviding a method for reaction to release the C-terminal amino acidssuccessively, with use of which method, when a reaction mechanism viaformation of oxazolone ring structure as explained above is used torelease the C-terminal amino acids from the peptide, undesired sidereactions such as cleavage of peptide bond somewhere along the peptidechain can be easily suppressed and further said chemical treatmentitself can be carried out under widely applicable conditions. Morespecifically, the aim of the present invention is to provide a methodfor analysis of C-terminal amino acid sequence of peptide, by using anovel means for reaction of releasing the C-terminal amino acidssuccessively, which means can avoid side reactions such as cleavage inthe middle of peptide, when successively releasing the C-terminal aminoacids, and can carry out the chemical treatment itself therefor in mildtemperature conditions near room temperature that do not need anyprecise temperature control with heating or cooling. Furthermore, thepresent invention ultimately aims at wide application of such a methodfor analysis of C-terminal amino acid sequence of peptide and, in moreparticular, aims at providing a kit for treatment of releasing theC-terminal amino acids successively according to said novel means forreaction of releasing the C-terminal amino acids from the peptide, whichis exclusively used in the analysis method of the present invention.

The present inventors made an intensive study and examinationcontinually in order to solve the above-mentioned problems. As a result,it was concluded that the undesired reactions seen in the case of themethod, where a high concentration aqueous solution of aperfluoroalkanoic acid is used to allow a vapor of the perfluoroalkanoicacid therefrom to act on a dried peptide under heating up conditions,for example, at 90° C., occur because the vapor of a perfluoroalkanoicacid as well as water molecule, both vaporized from the highconcentration aqueous solution of the perfluoroalkanoic acid, arepresent in the reaction system, for example, at the serine residue[NH—CH(CH₂OH)—CO—] in the peptide, the N,O-acyl rearrangement reactionbetween the amino group (—NH—) on its a-position and the hydroxy group(—OH) on its β-position is promoted under said heating conditions andthe hydrolysis of the ester formed thereby is also advanced by the helpof water molecules co-existing in the reaction system. Meanwhile, in thecase of the method, where an acetonitrile solution of aperfluoroalkanoic acid anhydride is used to allow a vapor of theperfluoroalkanoic acid anhydride therefrom to act on a dried peptideunder cooling down conditions, for example, at −18° C., it has beenconfirmed that although there is no water molecule in the reactionsystem, such a high reactivity of the perfluoroalkanoic acid anhydrideper se invites a rapid increase in the frequency of undesired sidereactions relative to rising up of the treatment temperature.

Based on the above finding, the present inventors searched such reactionconditions as, without using any water solvent working as source forfeeding water molecules to the reaction system and further without usingany reagent with such high reactivity as perfluoroalkanoic acidanhydride, an oxazolone ring structure can be formed from the C-terminalamino acids of peptide, as an reaction intermediate and then reaction ofselectively releasing the C-terminal amino acid can be completed inassociation with cleavage of the oxazolone ring. As a result, it wasfound that with use of a mixture obtained by adding a small amount of aperfluoroalkanoic acid to an alkanoic acid anhydride, when theperfluoroalkanoic acid and alkanoic acid anhydride, both of vapor phase,supplied from the mixture are allowed to act on a dried peptide, even ata treatment temperature such as 60° C. or less, the formation ofoxazolone ring structure can be progressed, and subsequently followed bythe reaction of selectively releasing the C-terminal amino acidtherefrom, which is resulted from the cleavage of this oxazolone ring.It was also found that as the reactivity of alkanoic acid anhydride issignificantly mild as compared with a perfluoroalkanoic acid anhydride,even in the co-presence of the perfluoroalkanoic acid, it is far fromgiving rise to any cleavage in the middle of peptide. Specificallyexplaining, the alkanoic acid anhydride acts, in the co-presence of theperfluoroalkanoic acid, on the hydroxy group present on the serineresidue [—NH—CH(CH₂OH)—CO—] or threonine residue [—NH—CH(CH(CH₃)OH)—CO—]in the peptide to make progress preferentially in an 0-acylationreaction, which leads to inhibiting the N,O-acyl rearrangement reactioncompetitively. It was also found that an N-acylation reaction to theamino group of N-terminal proceeds simultaneously and there alsoproceed, for example, an N-acylation reaction to the amino group on theε-position of lysine residue [—NH—CH(CH₂CH₂CH₂CH₂NH₂)—CO—] and anO-acylation reaction to the phenolic hydroxy group of tyrosine reside[—NH—CH(CH₂—C₆H₄—OH)—CO—]. As a result, it was found that since thereactive functional groups such as hydroxy group or amino group on theside chain, which are involved in the rearrangement reaction such asN,O-acyl rearrangement reaction that initiates the cleavage in themiddle of peptide, undergo protection and modification, undesired sidereactions are avoided and, at a treatment temperature of, for example,60° C. or less, there selectively proceed only reactions wherein theoxazolone ring structure is formed as the intended reaction intermediatefrom the C-terminal amino acid, and subsequently followed by thereaction of releasing the C-terminal amino acid in association with thecleavage of the oxazolone ring. Based on the above findings, the presentinventors have been completed the present invention.

Hence, the method for analysis of C-terminal amino acid sequence ofpeptide according to the present invention is

-   -   a method for analyzing the C-terminal amino acid sequence of a        peptide to be examined, which method comprises steps of:    -   releasing the C-terminal amino acids in sequence from the        peptide to be examined by chemical means to prepare a mixture        containing a series of reaction products thereof,    -   subjecting the series of reaction products and the original        peptide to mass spectrometry to measure the decreases in        molecular weight associated with the successive release of the        C-terminal amino acid thereof, and    -   identifying a series of the amino acids removed successively,        based on a series of the decrease in the molecular weight        measured and arranging the amino acids identified from the        C-terminal to obtain the information of the C-terminal amino        acid sequence of the peptide,    -   wherein the technique of treatment used in the step of releasing        the C-terminal amino acids is a means comprising steps of:    -   allowing an alkanoic acid anhydride and a perfluoroalkanoic acid        both of vapor phase, which are supplied from a mixture        containing an alkanoic acid anhydride with a small amount of a        perfluoroalkanoic acid added thereto, to act on a dry sample of        the peptide to be examined in a dry atmosphere at a temperature        selected in a range of 15 to 60° C.; and carrying out the        release of the C-terminal amino acid in association with the        process that at the C-terminus of the peptide, the formation of        a 5-oxazolone structure represented by the following general        formula (III):        wherein R1 is a side chain of the C-terminal amino acid of the        peptide, and R2 is a side chain of the amino acid residue        positioned just before the C-terminal amino acid, is followed by        the cleavage of the 5-oxazolone ring.

Further, as the alkanoic acid anhydride contained in said mixtureobtained by adding a small amount of a perfluoroalkanoic acid to thealkanoic acid anhydride, there is preferably used a symmetric anhydrideof an alkanoic acid having 2 to 4 carbon atoms. Among others, as saidsymmetric acid anhydride, a symmetric anhydride of a linear-chainalkanoic acid having 2 to 4 carbon atoms is preferred, and inparticular, acetic anhydride is used suitably. Meanwhile, as saidperfluoroalkanoic acid, there is preferably used a perfluoroalkanoicacid of which pKa is within the range of 0.3 to 2.5. As theperfluoroalkanoic acid, there can be suitably used, for example, aperfluoroalkanoic acid having 2 to 4 carbon atoms, and among the rest, alinear-chain perfluoroalkanoic acid having 2 to 4 carbon atoms is moreadapted. In said mixture obtained by adding a small amount of theperfluoroalkanoic acid to the alkanoic acid anhydride, the content ofthe perfluoroalkanoic acid is selected desirably in a range of 1 to 20%by volume relative to the total volume of the alkanoic acid anhydrideand the perfluoroalkanoic acid.

In the treatment using said mixture obtained by adding a small amount ofthe perfluoroalkanoic acid to the alkanoic acid anhydride, said dryatmosphere is preferably a state in which oxygen as well as water hasbeen removed. In particular, the dry atmosphere is more preferablyachieved inside an airtight vessel by vacuuming up the atmosphere insideit. Additionally, in the treatment using the mixture obtained by addinga small amount of the perfluoroalkanoic acid to the alkanoic acidanhydride, the temperature is more preferably set at a temperatureselected in a range of 15 to 50° C.

In the analysis method of the present invention, in addition to the stepof the treatment using said mixture obtained by adding a small amount ofthe perfluoroalkanoic acid to the alkanoic acid anhydride, hydrolysistreatment comprising steps of:

-   -   applying, to the mixture containing a series of reaction        products, which is obtained in said step of releasing the        C-terminal amino acids successively, a post-treatment of        removing the residual alkanoic acid anhydride and        perfluoroalkanoic acid therein in a dry state,    -   then, feeding a basic, nitrogen-containing aromatic cyclic        compound or a tertiary amine compound and water molecule both of        vapor phase supplied by using an aqueous solution in which the        basic, nitrogen-containing aromatic cyclic compound or a        tertiary amine compound is dissolved,    -   allowing the water molecule to act on the reaction products of        peptide in the presence of said basic, nitrogen-containing        organic compound, and    -   after said treatment for hydrolysis, re-drying post-treatment        which is conducted by removing the basic, nitrogen-containing        organic compound and water molecule both remaining in the        mixture containing a series of the reaction products, followed        by drying.

Further in the analysis method of the present invention, in addition tothe step of said treatment using the mixture obtained by adding a smallamount of the perfluoroalkanoic acid to the alkanoic acid anhydride,

-   -   the method may be provided, prior to the step of releasing the        C-terminal amino acids successively, with an additional step for        a pre-treatment of applying, to the N-terminal amino group of        the peptide to be examined, N-acylation protection in advance        with use of an acyl group derived from the alkanoic acid        constituting said alkanoic acid anhydride.

The pretreatment step of applying N-acylation protection to theN-terminal amino group may be conducted by employing technique where theN-acylation for amino groups in the peptide is effected by allowing analkanoic acid anhydride and an alkanoic acid both of vapor phase, whichare supplied from a mixture obtained by adding a small amount of thealkanoic acid to the alkanoic acid anhydride, to act on a dried sampleof the peptide to be examined in a dry atmosphere at a temperatureselected in a range of 10 to 60° C. In such a case, as the alkanoic acidanhydride used in the pretreatment step of applying N-acylationprotection to the N-terminus and as the alkanoic acid anhydride used inthe step conducted thereafter of releasing the C-terminal amino acidssuccessively, used may be the same alkanoic acid anhydride.

The present invention also provides a process for preparing a mixturecontaining a series of reaction products obtainable by releasing theC-terminal amino acids successively from a target peptide with use ofchemical means, which is corresponding to the most characteristic stepcomprised in the above-mentioned method for analysis of C-terminal aminoacid sequence of peptide according to the present invention; that is,the process of the present invention for preparing a mixture containinga series of reaction products obtainable by releasing the C-terminalamino acids successively from a peptide with use of chemical means isdefined as

-   -   a process for preparation of a mixture containing a series of        reaction products obtainable by releasing the C-terminal amino        acids successively from a target peptide with use of chemical        means,    -   wherein the process is conducted to prepare the mixture        containing a series of reaction products obtainable by releasing        the C-terminal amino acids successively from the peptide by the        chemical means comprising steps of:    -   allowing an alkanoic acid anhydride and a perfluoroalkanoic acid        both of vapor phase, which are supplied from a mixture of the        alkanoic acid anhydride with a small amount of the        perfluoroalkanoic acid added thereto, to act on a dried sample        of the target peptide in a dry atmosphere at a temperature        selected in a range of 15 to 60° C., and    -   carrying out the release of the C-terminal amino acid in        association with the process that at the C-terminus of the        peptide, the formation of a 5-oxazolone structure represented by        the following general formula (III):        wherein R1 is a side chain of the C-terminal amino acid of the        peptide, and R2 is a side chain of the amino acid residue        positioned just before said C-terminal amino acid, is followed        by the cleavage of the 5-oxazolone ring.

In addition, the present invention provides a kit for treatment that canbe exclusively used for said treatment technique to successively releaseC-terminal amino acids according to the present invention; that is, thekit of the present invention used for the treatment of releasing theC-terminal amino acids successively is defined as

-   -   a kit used for treatment of reaction to release the C-terminal        amino acids successively from a target peptide by chemical        means,    -   wherein the kit for treatment of releasing the C-terminal amino        acids successively is the kit being set up with a combination        of:    -   as a liquid reagent for the reaction of releasing the C-terminal        amino acids successively, at least a mixture obtained by a small        amount of a perfluoroalkanoic acid to an alkanoic acid        anhydride, or separately the alkanoic acid anhydride and the        perfluoroalkanoic acid in combination for preparation of the        mixture,    -   a sample container for holding a sample of the target peptide to        be treated therein, and    -   a reactor vessel which is provided with a liquid reagent-holding        system capable of reserving said liquid reagent therein and        capable of maintaining such a state that said liquid reagent        makes no direct contact with said peptide sample held in the        sample container and which has capacity to accommodate said        sample container inside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of process flow illustrating an example of thedetailed procedures employed in the treatment to successively releaseC-terminal amino acids from a peptide, according to the presentinvention.

FIG. 2 shows an example of the spectra observed in mass spectrometricanalysis of a mixture of reaction products, which are obtained bysuccessively releasing the C-terminal amino acids from angiotensin Ipeptide according to the treatment method of the present invention forreleasing C-terminal amino acids successively from the peptide,

FIG. 3 shows another example of the spectra observed in massspectrometric analysis of a mixture of reaction products, which areobtained by successively releasing the C-terminal amino acids fromangiotensin I peptide according to said treatment method of the presentinvention for releasing C-terminal amino acids successively from thepeptide.

FIG. 4 shows still other example of the spectra observed in massspectrometric analysis of a mixture of reaction products, which areobtained by successively releasing the C-terminal amino acids fromangiotensin I peptide according to the treatment method of the presentinvention for releasing C-terminal amino acids successively from thepeptide.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained in more detail below.

The method for analysis of C-terminal amino acid sequence of peptideaccording to the present invention is basically utilizing such atechnique comprising steps of releasing the C-terminal amino acids formthe peptide successively to prepare a series of reaction products withtruncated peptide chains relative to the peptide to be examined, andidentifying the amino acids released therefrom, based on the differencesbetween the series of the molecular weights for the reaction productsand the molecular weight for the original peptide. In more particular,the method uses mass spectrometry as means for measuring the molecularweights of the series of reaction products and the molecular weight ofthe original peptide, and among others, there is preferably used aTime-of-Flight type mass spectrometer, for example, a MALDI-TOF-MSsystem, which is very suitable for a measurement conducted under suchconditions as there takes place, in the step of ionization, no removalof fragments with atomic moieties from the amino acid residues composingthe peptide.

Meanwhile, the most characteristic feature in the analysis method of thepresent invention is that in the step of releasing the C-terminal aminoacids from the peptide successively, the method is using such atreatment comprising steps of

-   -   allowing an alkanoic acid anhydride and a perfluoroalkanoic acid        both of vapor phase, which are supplied from a mixture of the        alkanoic acid anhydride with a small amount of the        perfluoroalkanoic acid added thereto, to act on a dried sample        of the target peptide in a dry atmosphere at a temperature        selected in a range of 15 to 60° C., and    -   carrying out the release of the C-terminal amino acid in        association with the process that at the C-terminal of the        peptide, the formation of a 5-oxazolone structure represented by        the following general formula (III):        wherein R1 is a side chain of the C-terminal amino acid of the        peptide, and R2 is a side chain of the amino acid residue        positioned just before said C-terminal amino acid, is followed        by the cleavage of the 5-oxazolone ring.

The reaction of the formation of said 5-oxazolone ring is expressed as awhole by the following reaction scheme (I):

In particular, in the case of the process for selectively releasingC-terminal amino acid according to the present invention, at first, bychoice of the step of allowing an alkanoic acid anhydride and aperfluoroalkanoic acid both of vapor phase and supplied from a mixtureobtained by adding a small amount of a perfluoroalkanoic acid to analkanoic acid anhydride, to act on a dried peptide in a dry atmosphereat a temperature of a range of 15 to 60° C., at the stage of theketo-enol tautomerism represented by the following reaction formula(Ia):

the ratio for staying in the enol state is heightened by allowing theperfluoroalkanoic acid of vapor phase to function as a proton donortoward the dried peptide.

Then, an intramolecular ester bond is formed between the hydroxy groupexposed in the enol type and the C-terminal carboxy group to completethe 5-oxazolone ring-formation. On the other hand, in the case of theconventional method, the intramolecular ester bond is formed in a statein which a perfluoroalkanoic acid and water molecule, both of vaporphase, are generated from a high concentration aqueous solution of theperfluoroalkanoic acid by heating up to, for example, 90° C., andpresent together. It is expected that, in this esterification reactionas well, the perfluoroalkanoic acid of vapor phase maybe functions as aproton donor to induce the esterification reaction proceeding under anacid catalyst. However, since the reaction is carried out in asolvent-free solid phase, the reaction temperature is set high.Meanwhile, in the process for selectively releasing C-terminal aminoacid according to the present invention, an alkanoic acid anhydride isused as a reagent for activation of C-terminal carboxy group; thereoccurs conversion into an asymmetric acid anhydride as illustrated by,for example, the following reaction formula (Ib);

involved in the reaction. As a result, such a reaction can proceed undera mild temperature condition and the reaction temperature can beselected in a range of 15 to 60° C. Incidentally, such a reactiontemperature is selected preferably at around room temperature or in atemperature range slightly higher than room temperature, in moreparticular, more preferably in a range of 15 to 50° C.

Meanwhile, in the process for selectively releasing C-terminal aminoacid according to the present invention, as for the perfluoroalkanoicacid used therein, its proton-donating ability is used, and thus aperfluoroalkanoic acid of which pKa is within the range of 0.3 to 2.5 ispreferably used. In addition, since this perfluoroalkanoic acid needs tobe supplied to a dried peptide in a vapor phase, it is preferred thatthe perfluoroalkanoic acid being be superior in volatility is selectedso that a desired vapor pressure is obtained at said temperatureselected in a range of 15 to 60° C. From this standpoint as well, aperfluoroalkanoic acid having 2 to 4 carbon atoms is more suitable, anda liner-chain perfluoroalkanoic acid having 2 to 4 carbon atoms isfurther more adapted. Specifically, use of trifluoroacetic acid(CF₃COOH), pentafluoropropanoic acid (CF₃CF₂COOH) or heptafluorobutanoicacid (CF₃CF₂CF₂COOH) is more desired.

On the other hand, the alkanoic acid anhydride used as an activationreagent is consumed with the progress of the reaction; therefore, it isdesired to conduct the reaction while the vapor pressure of the alkanoicacid anhydride supplied in a vapor phase is maintained at a given level.Examples of the means adapted for the purpose include such a means thatthe reaction system is kept in a sealed state and thereby the vaporpressure of the alkanoic acid anhydride present in the system isstabilized. In more particular, exemplified is such a procedure in whicha liquid mixture obtained by adding a small amount of aperfluoroalkanoic acid to an alkanoic acid anhydride is placed in asealable reactor; the liquid mixture is once cooled to reduce its vaporpressure; in this state, the reactor inside is evacuated and then sealedoff; the alkanoic acid anhydride is vaporized in the reactor by heatingup to a reaction temperature. By employing such a procedure, there isanother advantage that the leakage of water into the reactor can beprevented. Further, when evacuation is conducted so that no oxygenremains in the reaction system, for example, the sulfur present inmethionine, which is included in the amino acid residues composing apeptide to be examined, can be prevented from oxidation by oxygen andconsequent change of its formula weight. In the method of the presentinvention based on the measurement of molecular weights, such preventionof oxidation is more preferred for achieving a higher accuracy.

Incidentally, for example, when the peptide to be examined contains aplurality of cysteines which are forming an —S—S— bond of its oxidizedtype between a cysteine of adjacent peptide or forming a —S—S— bondwithin one peptide molecule, an ordinary reduction treatment is appliedbeforehand to eliminate such a linkage and thereby the peptide isconverted into a peptide containing reduced forms of cysteines. Further,to the reduced form of cysteine present in a peptide is applied, for itsprotection, for example, carboxymethylation or pyridylethylation to thesulfanyl group (—SH) on its side chain.

Variety of alkanoic acid anhydrides, as long as they can produce anappropriate vapor pressure when heated to the temperature of reaction,is applicable as the alkanoic acid anhydride used therein. Meanwhile,there is preferred an alkanoic acid anhydride which gives a sufficientvapor pressure when the reaction temperature is selected in theabove-mentioned preferable range, for example, of 15 to 50° C.Therefore, a symmetric anhydride of an alkanoic acid having 2 to 4carbon atoms is used preferably. Among others, as the symmetric acidanhydride, a symmetric anhydride of a linear-chain alkanoic acidanhydride having 2 to 4 carbon atoms is used more preferably, and inparticular, a symmetric anhydride of a linear-chain alkanoic acid having2 carbon atoms, i.e. acetic acid is used appropriately. Since such analkanoic acid anhydride is used for the activation of C-terminal carboxygroup, the anhydride is preferred to give minimum steric hindrance, andthe above-mentioned acetic acid, etc. are very suitable in this respectas well.

In said reaction of releasing amino acids, the alkanoic acid anhydrideand the perfluoroalkanoic acid are allowed to act on a dried peptide inrespective vapor states. The reaction is conducted in a dry atmospherein order to avoid the hydrolysis of once-formed 5-oxazolone ring by thewater incoming from outside of the system and its reversion to originalstructure. In this view, the reaction is desired to be carried outgenerally in a sealed reactor. Incidentally, the mixture of the alkanoicacid anhydride and perfluoroalkanoic acid, initially fed into thereactor is, at room temperature, a liquid mixture wherein the alkanoicacid anhydride and perfluoroalkanoic acid are mixed uniformly. In thismixture containing the alkanoic acid anhydride with a small amount ofthe perfluoroalkanoic acid added thereto, the perfluoroalkanoic acidfunctioning as a catalyst is not consumed during the reaction inprinciple and therefore its content can be a small amount. Morespecifically explaining, the vapor of perfluoroalkanoic acid present inthe vapor phase, as compared with the vapor of alkanoic acid anhydrideco-existing, can be in a relatively low concentration. In other words,depending upon the kinds of the alkanoic acid anhydride andperfluoroalkanoic acid used, for instance, on the respective saturatedvapor pressures thereof at the reaction temperature, there isappropriately selected a liquid mixture having a mixing ratio which canachieve an intended partial pressure ratio (a concentration ratio invapor phase). The content of perfluoroalkanoic acid, in the mixturecontaining the alkanoic acid anhydride with a small amount of theperfluoroalkanoic acid added thereto, is desired to be selected, forexample, in a range of 1 to 20% by volume, preferably in a range of 3 to10% volume relative to the total volume of the alkanoic acid anhydrideand the perfluoroalkanoic acid.

In the process for selectively releasing C-terminal amino acid accordingto the present invention, it is concluded that, from the once-formed5-oxazolone ring, the separation of the C-terminal amino acid and theformation of the reaction intermediate for the next-stage may proceed,for instance, via such reaction as shown by the following reactionformula (II′):

as a result, successive release of C-terminal amino acids is advanced insuch way. Therefore, the reaction products obtained after the completionof such reactions are the mixture comprising, in addition to thosehaving a carboxy group exposed at the C-terminal, such as shown in theabove reaction formula (II), an intermediate product having the5-oxazolone ring structure and a form of reaction intermediate in whichits C-terminal is converted into the form of asymmetric acid anhydride.

The successive reaction used in the treatment step of releasingC-terminal amino acid selectively is constructed at least with two-stageelementary reactions, i.e. a stage of formation of 5-oxazolone ringstructure as illustrated by the reaction formula (Ib) and a stage ofseparation of C-terminal amino acid by the cleavage of 5-oxazolone ringstructure, as illustrated by the reaction formula (II′). Therefore, theoverall reaction rate depends upon the reaction rates of the two stages,but depends mainly upon the partial pressures (concentrations in vaporphase) of the alkanoic acid anhydride and perfluoroalkanoic acid usedtherefor as well as the reaction temperature therein. In addition, as aseries of reaction products are formed by successive reactions, themaximum length of C-terminal amino acid sequence removed, which can beattained in the series of reaction products obtained, becomes longer asthe treatment duration becomes longer. Hence, the treatment duration forthe treatment step of selectively releasing C-terminal amino acid insuch a successive manner needs to be appropriately chosen dependingmainly upon the partial pressures (concentrations in vapor phase) of thealkanoic acid anhydride and perfluoroalkanoic acid and the reactiontemperature employed therefor and also in view of the intended length ofthe C-terminal amino acid sequence to be analyzed.

It is possible to arrange additional treatment for hydrolysis at the endin order to convert the forms of reaction intermediate having no carboxygroup exposed at the C-terminal, such as illustrated in the abovereaction formula (II′), formed during the treatment step of selectivelyreleasing C-terminal amino acid in such a successive manner, into a formhaving a exposed carboxy group exposed at the C-terminal. Hence, in theprocess of the present invention for releasing C-terminal amino acidsselectively, it is preferred to provide, in addition to said step of thetreatment with use of a mixture obtained by adding a small amount of aperfluoroalkanoic acid to an alkanoic acid anhydride, the process withadditional steps for hydrolysis treatment comprising steps of:

-   -   applying, to the mixture containing a series of reaction        products, which is obtained in said step of releasing the        C-terminal amino acids successively, a post-treatment of        removing the residual alkanoic acid anhydride and        perfluoroalkanoic acid therein in a dry state,    -   then, feeding a basic, nitrogen-containing aromatic cyclic        compound or a tertiary amine compound and water molecule both of        vapor phase supplied by using an aqueous solution in which the        basic, nitrogen-containing aromatic cyclic compound or tertiary        amine compound is dissolved,    -   allowing the water molecule to act on the reaction products of        peptide in the presence of said basic, nitrogen-containing        organic compound, and    -   after said treatment for hydrolysis, re-drying post-treatment        which is conducted by removing the basic, nitrogen-containing        organic compound and water molecule both remaining in the        mixture containing a series of the reaction products, followed        by drying. By application of this post-treatment, the reaction        products come to take forms having a carboxy group exposed at        the C-terminal. These forms give major peaks in the analysis by        mass spectrometry conducted thereafter, which makes easier the        operation for identification of peaks with the molecular weights        corresponding to a series of reaction products in the light of        the peak intensities thereof.

The basic, nitrogen-containing aromatic cyclic compound or tertiaryamine compound both of vapor phase has no ability to react with, forexample, products remaining such a form in which its C-terminal has beenturned into an asymmetric acid anhydride or to form any amide bondtherewith; and further, the basic, nitrogen-containing aromatic cycliccompound or tertiary amine can be made into a uniform solution whenpreparing an aqueous solution thereof; which features are preferably fitto use for the treatment of hydrolysis. As the basic,nitrogen-containing aromatic cyclic compound which can be used, there ispreferred a monocyclic nitrogen-containing aromatic compound which cangive an appropriate vapor pressure, and, for example, pyridine can bemore suitably used. As the tertiary amine compound which can be used,there is preferred one having the same weak basicity as shown bypyridine and, for instance, such as DMAE [(CH₃)₂N—CH₂CH₂OH) can besuitably used. When, for example, pyridine is used, the pyridine contentis preferably selected in a range of 5 to 15% by volume, in moreparticular at 10% by volume relative to the whole volume of the aqueoussolution thereof. When (dimethylamino) ethanol (DMAE) is used, the DMAEcontent is preferably selected in a range of 1 to 20% by volume, in moreparticular at 10% by volume relative to the whole volume of the aqueoussolution thereof.

The monocyclic nitrogen-containing aromatic compound or tertiary aminecompound is allowed to act on a dried mixed sample containing thereaction products, in the vapor state together with water molecule. Inthis post-treatment, the reaction as well is desired to be conducted ina sealed reactor. In said post-treatment, since water molecule is used,its vapor pressure needs to be set at a certain level or higher.Therefore, the treatment temperature is desirably chosen, for example,from a temperature of 60° C. or more but, when the mechanical strengthof the reactor is taken into consideration, in a range of 100° C. orlower. In order to complete the treatment for hydrolysis quickly, such atemperature of 100° C. or slightly lower is desired to be selected.

Moreover, in the process for selectively releasing C-terminal amino acidaccording to the present invention, in addition to the step of thetreatment using a mixture obtained-by adding a small amount of aperfluoroalkanoic acid to an alkanoic acid anhydride, the process may beprovided, prior to said step of releasing the C-terminal amino acidssuccessively, with a step for a pre-treatment of applying, to theN-terminal amino group of the peptide to be examined, N-acylationprotection in advance with use of an acyl group derived from thealkanoic acid constituting said alkanoic acid anhydride. Specificallyexplaining, in said step of the treatment using a mixture obtained byadding a small amount of a perfluoroalkanoic acid to an alkanoic acidanhydride, a reaction intermediate wherein the C-terminal carboxy groupof peptide is activated, is presumed to be formed. When this reactionintermediate would react with the N-terminal amino group of an adjacentpeptide to form an amide bond therewith, no intended reaction productwith truncated peptide chain is obtained. Since the reaction per se iscarried out in a solid phase, frequency of such accidental side reactionis not so high. However, N-acylation is desirably applied beforehand toprotection against the side reaction.

In addition, during said treatment using a mixture obtained by adding asmall amount of a perfluoroalkanoic acid to an alkanoic acid anhydride,the N-terminal amino group of peptide ordinarily undergoes N-acylationby the alkanoic acid anhydride, and accordingly N-acylation protectiontakes place in the reaction system; nevertheless, it is more desired toconduct the pretreatment aiming at N-acylation protection.

The pretreatment step of applying N-acylation protection to theN-terminal amino group may be conducted by employing technique where theN-acylation for amino groups in the peptide is effected by allowing analkanoic acid anhydride and an alkanoic acid both of vapor phase, whichare supplied from a mixture obtained by adding a small amount of thealkanoic acid to the alkanoic acid anhydride, to act on a dried sampleof the peptide to be examined in a dry atmosphere at a temperatureselected in a range of 10 to 60° C. In such a case, it is preferred thatthe alkanoic acid anhydride used in the pretreatment step of applyingN-acylation protection to the N-terminal and as the alkanoic acidanhydride used in the step conducted thereafter of releasing theC-terminal amino acids successively, used is the same alkanoic acidanhydride. Specifically explaining, in the pretreatment reaction forapplying N-acylation protection to the N-terminal amino group, thereaction is allowed to proceed by supplying, to the dried peptidesample, the alkanoic acid anhydride and alkanoic acid both in a vaporstate; therefore, in order to obtain appropriate vapor pressuresthereof, there can be suitably used the same alkanoic acid anhydride asthe alkanoic acid anhydride used in the step conducted thereafter, ofreleasing the C-terminal amino acids successively. In addition, sincethe reactivity of the alkanoic acid anhydride is too low to causeundesired side reactions such as peptide cleavage, in a dry atmosphereat a temperature of a range of 10 to 60° C. and further since thealkanoic acid to be used together is strikingly inferior in acidcatalytic activity to perfluoroalkanoic acid, the combination canprovide N-acylation protection to N-terminal amino group in thepretreatment, with much less possibility to invite undesired sidereactions.

Additionally, in the step of N-acylation protection to the N-terminalamino group of peptide, N-acylation protection proceeds simultaneouslyalso to the ε-amino group on the side chain of the lysine residuepresent in the peptide. Furthermore, O-acylation proceeds also to thehydroxyl groups on the side chain of the serine and threonine residespresent in the peptide, and thereby protection thereof is made. Besides,the phenolic hydroxyl group on the side chain of the tyrosine residuepresent in the peptide also undergoes partially O-acylation although itsreactivity is varied. As a result of the pretreatment step wherein aplurality of these acylation protections take place as well, the aminogroup on the side chain of lysine residue and the hydroxyl group on theside chain of serine and threonine residues are all modified with theprotection group, and are no longer able to take part in undesired sidereactions. It is generally preferred from this standpoint as well thatthe pretreatment step of application of the N-acylation protection tothe N-terminal amino group of peptide is conducted in advance.

Incidentally, in the combination use of the alkanoic acid anhydride andalkanoic acid in the pretreatment step, there is substantially no fearof undesired side reactions, for example, such as cleavage in the middleof peptide. However, the pretreatment temperature is preferred to beselected in a range of 10 to 60° C., more preferably at around roomtemperature or a temperature range slightly higher than roomtemperature, and in more particular in a range of 15 to 50° C. On theother hand, the content of the alkanoic acid in the mixture obtained byadding a small amount of the alkanoic acid to the alkanoic acidanhydride is preferred to be selected in a range of 2 to 10% by volume,specifically at 5% by volume relative to the total volume of thealkanoic acid anhydride and alkanoic acid.

Besides, the procedure for reaction in the pretreatment step ispreferably conducted in very similar manner to the above-mentionedprocedure for the step of selectively releasing C-terminal amino acids.That is, the above-mentioned procedure for operation and conditionssuitable to the step for selectively releasing C-terminal amino acidsare also adapted to said reaction step in the pretreatment step.Incidentally, the rate of N-acylation reaction in the pretreatment stepdepends upon the partial pressures (concentrations in vapor phase) ofthe alkanoic acid anhydride and alkanoic acid used as well as thereaction temperature; therefore, the reaction time of the pretreatmentstep is desired to be selected appropriately depending mainly upon thereaction temperature. When the reaction temperature is selected at, forexample, 50° C., the reaction time is selected to be within one hour,for example, for 30 minutes, whereby the N-acylation to the N-terminalamino group of peptide can be completed. In such a case, addition ofpyridine of catalytic amount, for example, 0.1 to 1.0% by volumerelative to the total volume of the alkanoic acid anhydride and alkanoicacid is more preferred in order to enhance the acylation reaction withuse of the alkanoic acid anhydride and the alkanoic acid. Since thispyridine base functions as a proton receptor, for example, removal ofprotons to be eliminated in association with the acylation to aminogroup is made more swiftly.

Further preferably, the process for selectively releasing C-terminalamino acids according to the present invention is carried out in such amode in which the pretreatment step, the reaction step of selectivelyreleasing C-terminal amino acids and the post-treatment step are allcomprised. An example of such a flow pattern of steps is illustrated inFIG. 1. A drying-up operation is conducted in said flow when each stephas been completed, so that the reagents used in each step do not remainin the peptide sample. This drying-up operation is generally conductedby vacuum distillation, and thereby the C-terminal amino acids releasedthat are by-products in said reaction can be removed as well, in somecases. The flow pattern of steps of FIG. 1 illustrates an examplewherein acetic anhydride of high availability in a very high purity isutilized as the alkanoic acid anhydride used therein.

On the other hand, in the flow pattern of steps illustrated in FIG. 1,as for the treatment duration in the reaction step of selectivelyreleasing C-terminal amino acids, illustrated is a range of treatmenttime which is selected depending upon the proportions of the aceticanhydride and fluoroalkanoic acid used and the treatment temperatureemployed, for model case where the length of amino acids of theC-terminal amino acid sequence to be truncated in said step are intendedto be ten odd amino acids as maximum case and 3 amino acids as minimumcase. In general, when the proportion of the fluoroalkanoic acid islarger and the treatment temperature is higher, the reaction rate ishigher, and thereby it is possible to prepare a series of reactionproducts that have attained the maximum of truncated length of aminoacid sequence, which is set as the goal, in shorter treatment duration.

Furthermore, in the pretreatment step, N-acetylation to N-terminal aminogroup of peptide is carried out by using acetic anhydride and aceticacid both of vapor phase. Even in the case of such combination of aceticanhydride and acetic acid, there is, in some cases, a fear, maybe verysmall, that the activation reaction to C-terminal carboxy groupexpressed by the above-shown reaction formula (Ia) and the side reactioncaused thereby take place. In order to suppress such side reaction, asmall amount of pyridine vapor can be allowed to co-exist to form a weakaddition salt between the pyridine base and the C-terminal carboxy groupof peptide, which may provide protection effect against the occurrenceof the undesired side reaction. Such protection of addition saltformation type undergoes easy deprotection by conducting a drying-upoperation upon completion of the pretreatment step to distil off thepyridine base under vacuum, and no problem occurs in the next reactionstep of selectively releasing C-terminal amino acids. From thesestandpoints, it is preferred to add, for the protection of addition saltformation type, a small amount of a nitrogen-containing heterocyclicaromatic compound which can be easily distilled off under reducedpressure and has a weak basicity, such as pyridine. Further, since theprotection of addition salt formation type possesses protective functionalso for the carboxy group on the side chain of amino acid, it caneffectively prevent even the undesired side reaction that is originatedfrom the carboxy group on the side chain of amino acid coincidently.

In the method of the present invention for analysis of C-terminal aminoacid sequence of peptide, the molecular weights of the series ofreaction products prepared by successively releasing C-terminal aminoacids and the molecular weight of the original peptide are determined byconsulting measured data by mass spectrometry and there are identifiedamino acids corresponding to the differences in the molecular weightsthereof. Therefore, it is generally desired that the original peptideremains in the mixture subjected to the measurement by massspectrometry, in such an amount as to enable the determination of itsmolecular weight.

Specifically explaining, the method of the present invention foranalysis of the C-terminal amino acid sequence of peptide may be appliedto such a case where maximum length analyzed for the C-terminal aminoacid sequence is as long as ten and odd amino acids. With respect to thecontents of the series of reaction products of which sorts reach, asmaximum case, correspondingly into the number of ten and odd, thecontent of the minimum content reaction product is desired at least tobe not smaller than about {fraction (1/10)} of the content of themaximum content reaction product. In addition, the remaining amount ofthe original peptide as well is desired at least to be not smaller thanabout {fraction (1/10)} of the content of the maximum content reactionproduct. Meanwhile, the required information of C-terminal amino acidsequence is within 10 amino acids in many cases and, when selecting thetreatment time in which about 10 amino acids can be released, theabove-mentioned requirements regarding the contents can be satisfied.

Meanwhile, mass spectrometry is used for the measurement of molecularweight. The measurement is more suitably conducted using a massspectrometer equipped with such means for ionization that is welloperated under such conditions as to suppress the fragmentationresulting in detachment of part of atomic moieties from the amino acidresidues comprising the peptide, in the ionization step therein.Furthermore, as a peptide or the like has a high molecular weight, it ispreferred to use a Time-of-Flight type mass spectrometer, for instance,such as MALDI-TOF-MS system, which is suitable for measurement in such ahigh molecular weight range. However, even when such a type of massspectrometer is used, there is an upper limit as to the molecular weightallowing for effective ionization, and therefore it is desired that themaximum amino acids of a peptide that is possibly subjected tomeasurement does not exceed the limit of 20 to 30 amino acids. Inaddition, amino acids corresponding are identified based on the measureddifferences in molecular weight; therefore, in order to distinguish twoamino acid residues giving a formula weight difference of 1, forinstance such as Asn vs Asp, or Gln vs Glu, from each other at a highprecision, the molecular weight of the longest peptide, i.e. the peptidewith no release of C-terminal amino acid therefrom that is used as adatum point, is preferably in a range of no more than 3,000, morepreferably in a range of no more than 2,000. When reduced to aminoacids, it is preferred that its length is 30 amino acids at longest,more preferably in a range of no more than 20 amino acids.

When the method of the present invention for analysis of C-terminalamino acid sequence of peptide is applied to a peptide having length ofamino acids far more than the limit mentioned above, e.g. a protein, itis desired to conduct, prior to carrying out mass spectrometry,treatment for specific cleavage of the peptide is carried out by using,for example, a protease having a specificity for the cleavage site ofamino acid sequence, to allow the C-terminal peptide fragment obtainedto have length of amino acids within the above-mentioned range. That is,when the same treatment for site-specific cleavage is applied to boththe original peptide and a series of the reaction products preparedtherefrom, the resulting C-terminal peptide fragments are a series ofpeptide fragments which has the same N-terminal amino acid butdifference in C-terminal amino acid thereof. By using a mixturecomprising such series of peptide fragments to identify their molecularweights by means of mass spectrometry, the method of the presentinvention for analysis of C-terminal amino acid sequence of peptide canbe utilized.

However, when the present analysis method is applied to a long peptidesuch as a protein, in such a case that, in the long peptide, —S—S— bondbetween cysteine residues is formed owing to protein folding, it isnecessary to beforehand reduce the —S—S— bond to eliminate the bridgingbetween cysteine residues; further, the reduced form of cysteine ismodified by carboxymethylation or the like to protect the sulfanyl group(—SH) on the side chain. Also, in the region constituting a secondarystructure such as α-helix in association with the protein folding, thecarbonyl group (C═O) and imino group (—NH—) of amino acid residueconstituting the amide bond are in the state forming intramolecularhydrogen bond. When being held in such state forming the intramolecularhydrogen bond, the progress of the reaction used in the presentinvention may be suppressed. In view of this, it is desired that peptideis in advance treated to convert into the state where no secondarystructure is constructed at least in the C-terminal portion thereof, forexample, being treated for de-folding, and thereafter, drying of suchpeptide sample is conducted, and then the above-mentioned chemicaltreatment is applied thereto. When peptide is in the shape being treatedfor de-folding, for example, in such a case that after a series steps ofchemical treatments have been completed, site-specific cleavage of thepeptide using a protease or the like is needed prior to step of analysisin mass spectrometry, it has such an advantage that the C-terminalpeptide fragments obtained therein are generally easy to separate.

In the method of the present invention for analysis of C-terminal aminoacid sequence of peptide, the amino acids released successively areidentified based on the differences in molecular weight. Therefore,distinction between leucine (Leu) residue and isoleucine (Ile) residueboth having the same formula weight is impossible in principle, which isthe same as in the conventional method for analysis of C-terminal aminoacid sequence using mass spectrometry. On the other hand, in thereaction for releasing C-terminal amino acid, conversion of amide bondinto enol from and subsequent formation of 5-oxazolone ring structureare essential as shown in the reaction formula (Ib), and thus no furtherreaction for releasing proceeds when cyclic amino acid proline (Pro), inwhich any imino group (—NH—) forming amide bond together with carbonylgroup (C═O) is not present, has come to be the C-terminal amino acid. Inother words, by confirming that there occurs no further elimination ofC-terminal amino acid even when treatment duration is prolonged, it ispossible to predict that the amino acid residue that is main factor forsuch arrest is cyclic amino acid proline (Pro).

The process of the present invention for preparing a mixture comprisinga series of reaction products obtainable by releasing the C-terminalamino acids successively from the peptide with use of chemical meanscorresponds to the technique used in the reaction step of releasingC-terminal amino acid selectively for the method of the presentinvention for analysis of C-terminal amino acid sequence of peptideexplained above. Therefore, the preferred mode for carrying out theprocess is the same as described previously. This technique can beapplied not only for a linear peptide to prepare a sample used fordetermination of C-terminal amino acid sequence thereof but also for acyclic peptide to prepare a sample used for determining amino acidsequence thereof, wherein by ring-opening, the cyclic peptide is inadvance converted into form of linear peptide, which is subjected todetermination of its C-terminal amino acid sequence. Specificallyexplaining, various microorganisms for example, produce cyclic peptidetype compounds having biological activities, and said technique can beapplied for preparation of a sample for determination of the structuresof such compounds.

Even in said process of the present invention for preparing a mixturecontaining a series of reaction products obtainable by releasing theC-terminal amino acids successively from the peptide with use ofchemical means, for example, when there occur N,O-acyl rearrangement forthe hydroxy groups present on the serine residue [—NH—CH(CH₂OH)—CO—] andthreonine residue [—NH—CH(CH(CH₃)OH)—CO—] in the peptide, whichsubsequently results in branching, the ester bond therein, as comparedwith amide bond, undergoes cleavage more easily. However, owing to theaction of alkanoic acid anhydride and perfluoroalkanoic acid both ofvapor phase, O-alkanoylation to the hydroxy group on their side chainproceeds preferentially, which attains effectively competitiveinhibition against the N,O-acyl rearrangement. Incidentally, when thepost-treatment step is carried out, the ester bond to alcoholic hydroxygroup, as compared with the ester bond to phenolic hydroxy group,undergoes hydrolysis more quickly, and accordingly there remain, at highselectivities in the reaction products obtained finally, onlyN-alkanoylation to N-terminal amino group, N-alkanoylation to the aminogroup on the ε-position of lysine residue [—NH—CH(CH₂CH₂CH₂CH₂NH₂)—CO—]and, in some cases, also O-alkanoylation to the phenolic hydroxy groupof tyrosine residue [—NH—CH(CH₂—C₆H₄—OH)—CO—].

For example, when numbers of acetylated forms of serine residue andthreonine residue are included in the reaction products obtainedfinally, the molecular weight differences between such multi-acetylatedproduct and deacetylated product are aligned in the integral times offormula weight 42, specifically 84, 126 and 168 are close to the formulaweight 87 of serine residue [—NH—CH(CH₂OH)—CO—], the formula weight 128of glutamine residue [—NH—CH(CH₂CH₂—CONH₂)—CO—] or the formula weight129 of glutamic acid residue [—NH—CH(CH₂CH₂—COOH)—CO—] and the formulaweight 170 of N-acetyllysine residue [—NH—CH(CH₂CH₂CH₂CH₂NH—COCH₃)—CO—],respectively. Therefore, there is some fear that the peak for themulti-acetylated products may be mistaken as main peaks, and thedeacetylated products may be mis-assigned as derived products therefromwith the release of such an amino acid. However, actual measurement ismade in such an analytical precision that the distinction betweenglutamine residue and glutamic acid residue, of which the formulaweights differ only 1, is possible; since the formula weight differencerelated to the difference in number of remaining acetyl groups differsfrom the formula weight of amino acid residue showing a similar formulaat least with the Formula weight difference of 2 to 3; the possibilityof such mis-assignment mentioned above is not high in many cases.However, it is preferred to carry out the post-treatment to eliminatesuch undesired remains of the alkanoyl groups.

The kit of the present invention used for treatment to release theC-terminal amino acids successively is a kit being set up with acombination of a reactor vessel and a set of reagents usable under thereaction conditions suitable for said reactor vessel, which isapplicable to the reactions used exclusively in the aforementionedprocess of the present invention for preparing a mixture containing aseries of reaction products obtainable by releasing the C-terminal aminoacids successively from a peptide with use of chemical means. The set ofreagents for said reactions include, as a liquid reagent for thereaction of releasing the C-terminal amino acids successively, at leasta mixture obtained by a small amount of a perfluoroalkanoic acid to analkanoic acid anhydride, or separately the alkanoic acid anhydride andthe perfluoroalkanoic acid in combination for preparation of themixture. Since the same type of reactor can be used also in thepretreatment step as well as the post-treatment step explained above,the kit may be set up to further comprise the reagents used in thepretreatment step and the post-treatment step therein. It is preferablefor the kit to select the compositions and use amounts of these reagentsbeing adapted to the reaction conditions explained above.

With respect to the sample container for holding a sample of targetpeptide to be treated, since a solution containing the peptide sample isfed therein, and then treatment for drying up is conducted, andsubjected thereafter to an intended treatment(s), the sample containercan be formed in shape of a micro vial type used in treatment offreeze-drying or of a multi-well plate type used in simultaneoushandling of a plurality of peptide samples.

The reactor vessel is provided with a liquid reagent-holding system thatis capable of reserving the liquid reagent for said reaction or each ofthe liquid reagents combined in the component kit thereof respectively,capable of feeding the liquid reagents for said reactions at given ratesto the peptide sample held in said sample container, and capable ofmaintaining such a state that their direct contact with each other isavoided, and the reactor vessel has capacity to accommodate said samplecontainer inside. Preferably, the reactor vessel is designed in such aform that the inside can be evacuated, the liquid reagents remainingtherein after the completion of the reaction can be distilled off underreduced pressure, and the structure can be made gas-tight during thereaction In addition, the reactor vessel is required to be made of sucha material that, when the vapor of the reagent is generated in thereactor vessel, no reaction takes place between the reagent and the wallof the vessel. Therefore, there is suitably used such a vessel formed byusing glass material that is widely used for reactor in chemicalreactions. For the cocks used in a sealed-state operation, cocks made ofsuch a material as Teflon® or the like is used suitably.

EXAMPLES

The present invention is described specifically below by way ofExamples. These Examples are examples of the best mode for carrying outthe present invention; however, the present invention is in no wayrestricted by such specific embodiments illustrated thereby.

Example 1

In order to verify the usefulness of the method for analysis of theC-terminal amino acid sequence of peptide according to the presentinvention, analysis of C-terminal amino acid sequence was conducted forhuman angiotensin I, which is a peptide comprising 10 amino acids.

With respect to human angiotensin I, which is the peptide to be examinedin the present Example, its amino acid sequence is already known to beAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu. Using this peptide, theprecision of identification for the C-terminal amino acid sequenceanalyzed by means of the analysis method according to the presentinvention was verified.

(Pretreatment Operation)

First, a peptide solution containing 10 pmol of commercially availablehuman angiotensin I is fed into a micro vial and subjected to afreeze-drying treatment. The vial containing the dried peptide samplewas set in a glass-made reactor of air-tight test tube type with fittingstopper, having an evacuation port equipped with a Teflon-made cockvalve for sealing. Separately, a given amount of the following liquidreagent is placed in the glass-made reactor.

As the reagent for pretreatment, there is used 300 μl of aceticanhydride containing 5% by volume of acetic acid. After the vialcontaining the dried peptide sample has been set up in the glass-madereactor, the reactor inside is evacuated under cooling condition andthen sealed in an air-tight state.

The whole reactor of air-tight state is kept at 50° C. for 1 hour toallow acetic anhydride and acetic acid both of vapor phase, suppliedfrom the liquid reagent in the reactor, to act on the dried peptidesample. By allowing acetic anhydride in the co-presence of acetic acidas an acylation reagent to act on the dried peptide sample, selectiveacetylation to the N-terminal amino group of the peptide proceeds. As aresult, the peptide is converted into N-acetylated human angiotensin I.After such pretreatment has been completed, the unreacted aceticanhydride, acetic acid, etc. remaining in the reactor is distilled offunder reduced pressure and the N-acetylated human angiotensin I obtainedis dried.

(Operation for Releasing C-Terminal Amino Acid)

Next, in a state that the vial holding the dried sample of N-acetylatedhuman angiotensin I prepared is set similarly in the glass-made reactorof air-tight test tube type with fitting stopper, a given amount of thefollowing liquid reagent is placed anew in the glass-made reactor.

As a liquid reagent for the reaction of selectively releasing C-terminalamino acid, 300 μl of acetic anhydride containing 5% by volume oftrifluoroacetic acid is used. After the vial containing the dried samplehas been set up-in the glass-made reactor, the reactor inside isevacuated under cooling condition and then sealed in an air-tight state.

The whole reactor of air-tight state is kept at 40° C. for 16 hours toallow acetic anhydride and trifluoroacetic acid both of vapor phase,supplied from the liquid reagent in the reactor, to act on the driedsample. In this case, since acetic anhydride functions as an acylationreagent in the co-presence of trifluoroacetic acid, O-acetylationproceeds to the phenolic hydroxy group on the side chain of tyrosine(Tyr). Meanwhile, at the C-terminal of peptide, there proceed keto-enoltautomerism represented by the following reaction formula (Ia), promotedby trifluoroacetic acid functioning as a proton donor:

and conversion into asymmetric acid anhydride and formation of cyclicester, which occur by the action of acetic anhydride on C-terminalcarboxy group and are represented by the following reaction formula(Ib);

and whereby the C-terminal is once converted into a 5-oxazolonestructure. Further, the oxazolone ring is cleaved with the help ofacetic acid (CH₃COOH) by-produced in the above reaction or oftrifluoroacetic acid which is co-present, and the C-terminal amino acidis eliminated as N-acylated amino acid [Ac—NH—CH(R1)—COOH] and from theamino acid residue [—NH—CH(R2)—CO—] positioned just before theC-terminal acid, its terminal carboxy group comes to be exposed newly.Furthermore, a reaction to the next stage proceeds and the next aminoacid ahead that has been newly exposed at C-terminal is converted into a5-oxazolone structure as well.

Thus, leucine (Leu), histidine (His) and phenylalanine (Phe) aresuccessively released from the C-terminal of human angiotensin I toreach reaction product in which proline (Pro) is exposed at theC-terminal. Proline contains amino nitrogen in the ring structure;therefore, when a peptide bond is formed thereto, there occurs noketo-enol tautomerism represented by the above-shown reaction formula(Ia). Accordingly, the reaction can never progress in further conversioninto a 5-oxazolone structure represented by the reaction formula (Ib).As a result, under the conditions of allowing a vapor phase aceticanhydride to act in the co-presence of trifluoroacetic acid, there is noselective release of proline (Pro) positioned at the C-terminal That is,the reaction products obtained in the present Example are only threekinds of truncated peptides in which leucine (Leu), histidine (HiS) andphenylalanine (Phe) have been released successively. On the other hand,with respect to the C-terminals of said three kinds of truncatedpeptides, they are in the mixture state including those staying in the5-oxazolone structure, or being advanced even in conversion into anasymmetric acid anhydride therefrom, other than those being convertedinto carboxy group.

After the completion of the treatment for selective releasing C-terminalamino acids, the unreacted acetic anhydride, trifluoroacetic acid, etc.remaining in the reactor are distilled off under reduced pressure and amixture of the residual N-acetylated human angiotensin I and thereaction products obtained is dried.

(Post-Treatment Operation)

Next, in a state that the vial holding the dried sample of a mixture ofN-acetylated human angiotensin I and the obtained reaction products isset similarly in the glass-made reactor of air-tight test tube type withfitting stopper, a given amount of the following liquid reagent isplaced anew in the glass-made reactor.

As in the above mixture, the C-terminals of reaction products of peptideare in the mixture state including those staying in the 5-oxazolonestructure or being advanced even in conversion into an asymmetric acidanhydride therefrom, other than those being converted into carboxygroup, the post-treatment is a treatment mainly aiming to convert theminto the state where the C-terminals of the peptides all turned carboxygroups by applying treatment for hydrolysis to them. That is, an aqueoussolution of a basic, nitrogen-containing organic compound is used as aliquid reagent for post-treatment, and the basic, nitrogen-containingorganic compound and water molecule both of vapor phase, generated fromthe solution are allowed to act on said mixture which is driedbeforehand. In the present Example, 300 μl of an aqueous solutionholding 10% by volume of pyridine therein is used as the liquid reagentfor the post-treatment of hydrolysis; and after the vial containing thedried sample has been set in the glass-made reactor, the inside of thereacter is evacuated under cooling condition and then sealed in anair-tight state.

The whole reactor of air-tight state is heated at 100° C. for 30 minutesto allow the vapor-phase pyridine and water molecule, supplied from theliquid reagent in the reactor, to act on the dried sample. Theasymmetric acid anhydride and the 5-oxazolone structure undergohydrolysis by the action of water molecule in the co-presence ofpyridine base, whereby they are converted into a form having a carboxygroup at the C-terminal. Hydrolysis of ester bond proceeds also to theO-acetylated phenolic hydroxy group on the side chain of tyrosine (Tyr),whereby deacetylation (deprotection) thereof is made partially.Meanwhile, in N-acetylation, i.e. the acetyl group being substituted onN-terminal amino group, no hydrolysis of amide bond takes place underthe above-mentioned conditions. Therefore, the reaction productsobtained after the post-treatment are N-acetylated products in which theN-terminal of peptide is modified with the acetyl group. Consequently,in the reaction products, the N-terminal of peptide is modified with theacetyl group and the C-terminal has an exposed carboxy group; but theystay still in such state that those containing O-acetylation on the sidechain of the tyrosine (Tyr) are partially remained.

After the post-treatment has been completed, the residual watermolecule, pyridine, etc. in the reactor are distilled off under reducedpressure; and a mixture of N-acetylated human angiotensin I and thereaction products obtained after post-treatment is dried.

(Charactrization of Reaction Products Resulted After Post-Treatment)

The mixture of N-acetylated human angiotensin I and the reactionproducts resulted after post-treatment, prepared by conducting theseries of chemical treatments as explained above, is subjected to massspectrometry to measure the molecular weights of the individual peptidescontained therein.

In the present Example, using a Time-of-Flight type mass spectrometer,specifically, MALDI-TOF-MS system, there are conducted, on the driedmixture sample (for C1/3 analysis), measurement of the masses of mainion species, which are reflecting the molecular weights of individualpeptides, and their relative signal intensities, and subsequentlycomparison thereof. Mass spectrometry under the same conditions isconducted separately for the dried sample of N-acetylated humanangiotensin I obtained from the pretreatment alone, to determine themass of main ion species from N-acetylated human angiotensin I to beused as a reference.

FIG. 2 shows the mass spectrum measured for the mixture containingreaction products, which are treated for successively releasingC-terminal amino acids by said method of chemical treatment mentioned inthe present Example. The mass of the main ion species from N-acetylatedhuman angiotensin I is measured separately, referring to the measuredmass for said peak, there are identified the reaction productscorresponding to the important peaks measured in the mass spectrum ofFIG. 2. In Table 1 are shown the masses of the peaks measured, theirdifferences from the mass of the peak due to the original N-acetylatedhuman angiotensin I, and the amino acid residues, which are removed inindividual reaction products, identified based thereon, as well as theforms of individual reaction products. TABLE 1 Corresponding m/Z ΔmAssignment peptide structure 1380.7 +42.0 +CH₃CO Ac-DRVY(Ac)IHPFHL1338.7 — Ac-DRVYIHPFHL 1267.6 −71.1 −Leu, +CH₃CO Ac-DRVY(Ac)IHPFH 1224.6−114.1 −Leu Ac-DRVYIHPFH 1130.5 −208.2 −His.Leu, +CH₃CO Ac-DRVY(Ac)IHPF1088.5 −250.2 −His.Leu Ac-DRVYIHPF 983.5 −355.2 −Phe.His.Leu,Ac-DRVY(Ac)IHP +CH₃CO 941.5 −397.2 −Phe.His.Leu Ac-DRVYIHP

The peak due to the original N-acetylated human angiotensin I isaccompanied by a peak having a mass which is larger by 42. The latterpeak is judged to be due to those being modified with additional acetylgroup, i.e. those having the O-acetylation on the side chain of tyrosine(Tyr). Assuming that the remaining three peaks each accompanied by apeak having a mass which is larger by 42, are due to those products atC-terminal of which the amino acids are successively released, naturallyoccurring amino acid residues that will give rise to its deference inthe masses measured are identify. Since no further releasing proceedsafter the release of these three amino acids, the fourth amino acidresidue from C-terminal is concluded to be proline. Incidentally,leucine (Leu) and isoleucine (Ile) have the same formula weight andcannot be distinguished from each other in analysis by massspectrometry; however, in Table 1, they are expressed as leucine (Leu).

It is confirmed by the above verification experiment that by conductingthe treatment for selectively releasing C-terminal amino acids accordingto the present invention, a series of reaction products wherein theC-terminal amino acids have been successively removed from the originalpeptide, are obtained under mild treatment conditions and the C-terminalamino acid sequence of the peptide can be analyzed at a high accuracy.

Example 2

In this Example, it was verified that there occurs no progress of sidereaction that may result in serious problem even if the reactiontemperature varies slightly during the reaction of successivelyreleasing C-terminal amino acids, because mild treatment conditions areemployed in the treatment technique for selectively releasing C-terminalamino acids according to the present invention. In particular, thetreatment temperature employed for the reaction of releasing C-terminalamino acids, which is included in the series of chemical treatmentsdescribed in Example 1; was changed from 40° C. to 50° C., and thereaction products obtained in these two case were compared with eachother.

There were chosen the same procedure and conditions for each operationas the operational procedure and conditions described in Example 1, withthe except of said change in the temperature for the reaction ofreleasing by using acetic anhydride with 5% by volume of trifluoroaceticacid added threreto.

FIG. 3 shows the mass spectrum measured for the mixture comprisingreaction products, obtained by the treatment for successively releasingC-terminal amino acids in said way for chemical treatments as explainedin the present example. Summarized in Table 2 are the results ofassignment for reaction products corresponding to the important peaksmeasured in the mass spectrum of FIG. 3. Comparison between the spectrumof FIG. 3 and the spectrum of FIG. 2 confirms that even when thetemperature for treatment of releasing is increased from 40° C. to 50°C., there is no further progress in removal after three amino acids fromC-terminal has been removed and there occurs no side reaction such ascleavage in the middle of the peptide. TABLE 2 Corresponding m/Z ΔmAssignment peptide structure 1380.7 +42.0 +CH₃CO Ac-DRVY(Ac)IHPFHL1338.7 — Ac-DRVYIHPFHL 1265.7 −73.0 −Leu, +CH₃CO Ac-DRVY(Ac)IHPFH 1223.6−115.1 −Leu Ac-DRVYIHPFH 1131.6 −207.1 −His.Leu, +CH₃CO Ac-DRVY(Ac)IHPF1088.6 −250.1 −His.Leu Ac-DRVYIHPF 983.5 −355.2 −Phe.His.Leu,Ac-DRVY(Ac)IHP +CH₃CO 941.5 −397.2 −Phe.His.Leu Ac-DRVYIHP

Example 3

In said cases of Example 1 and Example 2, the pretreatment operation wascarried out in order to modify the N-terminal amino group of peptide forits protection. In the present Example, it was verified that no sidereaction causing serious troubles takes place even when the pretreatmentis not carried out, because, in the process for the chemical treatmentaccording to the present invention, at the step of carrying out thetreatment of successively releasing C-terminal amino acids, a peptidesample used is dried up in advance into a solid phase, and the reactionis promoted by supplying both acetic anhydride and trifluoroacetic acidin a vapor phase, which are utilized in the reaction.

Specifically explaining, the pretreatment described in Example 1 wasomitted, and the operation for the reaction of releasing C-terminalamino acids and the subsequent post-treatment operation were conductedto a dried sample of human angiotensin I. In this case, in the operationfor the reaction of releasing C-terminal amino acids, the treatmenttemperature was selected at 50° C. as in Example 2.

In the operation, as acetic anhydride and trifluoroacetic acid aresupplied in a vapor phase to give rise to a reaction, there proceedsimultaneously, in addition to the reaction of releasing C-terminalamino acids, N-acetylation to the N-terminal amino group of peptide andO-acetylation to the phenolic hydroxy group on the side chain oftyrosine. Further, there occur such by-products that have modificationsof trifluoroacetyl group to the N-terminal amino group of peptide and tothe phenolic hydroxy group on the side chain of tyrosine therein, whichis presumably attributable to such phenomena that when acting aceticanhydride and trifluoroacetic acid coincidently, the exchange reactionbetween these two may also occur.

In FIG. 4 is shown the mass spectrum observed for the mixture containingreaction products resulted from the treatment for successively releasingC-terminal amino acids conducted by said method for chemical treatmentsas explained in the present example. In Table 3 are summarized theresults of assignment for reaction products corresponding to theimportant peaks measured in the mass spectrum shown in FIG. 4.Comparison of the spectra of FIG. 4 and FIG. 3 confirms that, even whenno pretreatment is conducted, N-acetylation to the N-terminal aminogroup of peptide proceeds as well simultaneously, no further removaltakes place after the 3 amino acids from the C-terminal has beenremoved, and there is no side reaction such as cleavage in the middle ofthe peptide. Incidentally, the amide bonds for the N-terminal aminogroups being modified with trifluoroacetyl group are partiallyhydrolyzed in the post-treatment with far more ease than those beingmodified with acetyl group; therefore, there is observed an accessorypeak having a mass which is smaller by 42, corresponding to reactionproducts in which the N-terminal amino group is not N-acylated. That is,in Example 1 and Example 2, since modification by acetyl group is madeto the N-terminal amino gorup of peptide in the pretreatment step, thereis no reaction product in which the N-terminal amino group is modifiedwith a trifluoroacetyl group in place of an acetyl group; however, inthe present Example, there are also formed some portion of reactionproducts in which the N-terminal amino group are modified with atrifluoroacetyl group. In the spectrum of FIG. 4, there are indeedobserved collateral peaks attributed to the modification of N-terminalamino group with trifluoroacetyl group, having a mass which is larger by54 that of a peak due to that having the modification of N-terminalamino group with acetyl group. TABLE 3 Corresponding m/Z Δm Assignmentpeptide structure 1380.7 +42.0 +CH₃CO Ac-DRVY(Ac)IHPFHL 1338.7 —Ac-DRVYIHPFHL 1277.6 −61.1 −Leu+Δ(CF₃—CH₃) Tf-DRVYIHPFH 1265.7 −73.0−Leu, +CH₃CO Ac-DRVY(Ac)IHPFH 1223.6 −115.1 −Leu Ac-DRVYIHPFH 1183.6−155.1 −Leu−CH₃CO DRVYIHPFH 1088.6 −250.1 −His.Leu Ac-DRVYIHPF 1046.6−292.1 −His.Leu−CH₃CO DRVYIHPF 993.5 −345.2 −Phe.His.Leu, Tf-DRVYIHP+Δ(CF₃—CH₃) 983.5 −355.2 −Phe.His.Leu, Ac-DRVY(Ac)IHP +CH₃CO 941.5−397.2 −Phe.His.Leu Ac-DRVYIHP 899.5 −439.2 −Phe.His.Leu−CH₃CO DRVYIHP

Furthermore, it is clearly confirmed that, in addition to theN-acetylation to the N-terminal amino group of peptide, O-acetylation tothe phenolic hydroxy group on the side chain of tyrosine proceedssimultaneously. Meanwhile, as the reaction is practiced in a solidphase, there are effectively avoided such undesired side reactions thatthe activated reaction intermediates, for example asymmetric acidanhydride, formed in the reaction of successively releasing C-terminalamino acids, would act on the N-terminal amino group of peptide and thephenolic hydroxy group on the side chain of tyrosine. In contrast, theN-acetylation and O-acetylation effected by the acetic anhydride andtrifluoroacetic acid both supplied in a vapor phase progress rapidly. Asa result, it is confirmed that the reaction of successively releasingC-terminal amino acids proceeds in such state that the peptide has beenin situ modified for its protection.

Industrial Applicability

In the method for analysis of C-terminal amino acid sequence of peptideaccording to the present invention, as the means for successivelyreleasing the C-terminal amino acids of peptide, employed is suchtechnique comprising steps of allowing the alkanoic acid anhydride andperfluoroalkanoic acid both of vapor phase, supplied from a mixtureobtained by adding a small amount of an alkanoic acid anhydride to aperfluoroalkanoic acid, to act on a peptide to be examined of driedsolid state in a dry atmosphere at a temperature selected in a range of10 to 60° C., and and carrying out the release of the C-terminal aminoacid in association with the process that formation of a 5-oxazolonestructure thereof is followed by the cleavage of the 5-oxazolone ring toprepare a series of reaction products. In this technique, since thealkanoic acid anhydride used per se has a low reactivity, it is possibleto successively release the C-terminal amino acids of peptide under amild temperature condition such as a temperature selected in a range of15 to 60° C., preferably room temperature or a temperature slightlyhigher than that, for instance, a temperature selected in a range of 15to 50° C., without inviting undesired side reactions such as cleavage ofamide bond in the middle of peptide. In this connection, since there isno cleavage of amide bond in the middle of peptide, it is feasible toavoid mixing the peptide fragment resulting from said cleavage of amidebond and the reaction product originating from the peptide fragment inthe aimed reaction products obtained. Furthermore, with use of thereaction under such a mild condition, it is possible to attain moresuperior adjustment and control of the maximum length of the amino acidsto be cut off for the C-terminal amino acid sequencing. Therefore, inthe light of such merits as the excellent controllability and the mildreaction conditions, for instance the wide range of admittable variationin reaction temperature in the process for successively releasing theC-terminal amino acids of peptide, the method for analysis of theC-terminal amino acid sequence of peptide according to the presentinvention comes up to an analytical method having wide applicability.

1. A method for analyzing the C-terminal amino acid sequence of apeptide to be examined, which method comprises steps of: releasing theC-terminal amino acids in sequence from the peptide to be examined bychemical means to prepare a mixture containing a series of reactionproducts thereof, subjecting the series of reaction products and theoriginal peptide to mass spectrometry to measure the decreases inmolecular weight associated with the successive release of theC-terminal amino acid thereof, and identifying a series of the aminoacids removed successively, based on a series of the decrease in themolecular weight measured and arranging the amino acids identified fromthe C-terminal to obtain the information of the C-terminal amino acidsequence of the peptide, wherein the technique of treatment used in thestep of releasing the C-terminal amino acids is a means comprising stepsof: allowing an alkanoic acid anhydride and a perfluoroalkanoic acidboth of vapor phase, which are supplied from a mixture containing analkanoic acid anhydride with a small amount of a perfluoroalkanoic acidadded thereto, to act on a dry sample of the peptide to be examined in adry atmosphere at a temperature selected in a range of 15 to 60° C.; andcarrying out the release of the C-terminal amino acid in associationwith the process that at the C-terminal of the peptide, the formation ofa 5-oxazolone structure represented by the following general formula(III):

wherein R1 is a side chain of the C-terminal amino acid of the peptide,and R2 is a side chain of the amino acid residue positioned just beforethe C-terminal amino acid, is followed by the cleavage of the5-oxazolone ring.
 2. A method claimed in claim 1, wherein a symmetricanhydride of an alkanoic acid having 2 to 4 carbon atoms is used as thealkanoic acid anhydride contained in said mixture obtained by adding asmall amount of a perfluoroalkanoic acid to the alkanoic acid anhydride.3. A method claimed in claim 2, wherein a symmetric anhydride of alinear-chain alkanoic acid having 2 to 4 carbon atoms is used as saidsymmetric anhydride of an alkanoic acid having 2 to 4 carbon atoms.
 4. Amethod claimed in claim 1, wherein acetic anhydride is used as thealkanoic acid anhydride contained in said mixture obtained by adding asmall amount of a perfluoroalkanoic acid to the alkanoic acid anhydride.5. A method claimed in claim 1, wherein a perfluoroalkanoic acid ofwhich pKa is within the range of 0.3 to 2.5 is used as theperfluoroalkanoic acid contained in said mixture obtained by adding asmall amount of the perfluoroalkanoic acid to the alkanoic acidanhydride.
 6. A method claimed in claim 1, wherein a perfluoroalkanoicacid having 2 to 4 carbon atoms is used as the perfluoroalkanoic acidcontained in said mixture obtained by adding a small amount of theperfluoroalkanoic acid to the alkanoic acid anhydride.
 7. A methodclaimed in claim 6, wherein a linear-chain perfluoroalkanoic acid having2 to 4 carbon atoms is used as said perfluoroalkanoic acid having 2 to 4carbon atoms.
 8. A method claimed in claim 1, wherein in said mixtureobtained by adding a small amount of the perfluoroalkanoic acid to thealkanoic acid anhydride, the content of the perfluoroalkanoic acid isselected in a range of 1 to 20% by volume relative to the total volumeof the alkanoic acid anhydride and the perfluoroalkanoic acid.
 9. Amethod claimed in claim 1, wherein, in the treatment using said mixtureobtained by adding a small amount of the perfluoroalkanoic acid to thealkanoic acid anhydride, said dry atmosphere is a state in which oxygenas well as water has been removed.
 10. A method claimed in claim 9,wherein the dry atmosphere is achieved inside an airtight vessel byvacuuming up the atmosphere inside it.
 11. A method claimed in claim 1,wherein, in the treatment using the mixture obtained by adding a smallamount of the perfluoroalkanoic acid to the alkanoic acid anhydride, thetemperature is set at a temperature selected in a range of 15 to 50° C.12. A method claimed in claim 1, wherein, in addition to the step of thetreatment using said mixture obtained by adding a small amount of theperfluoroalkanoic acid to the alkanoic acid anhydride, the method isprovided with additional steps for hydrolysis treatment comprising stepsof: applying, to the mixture containing a series of reaction products,which is obtained in said step of releasing the C-terminal amino acidssuccessively, a post-treatment of removing the residual alkanoic acidanhydride and perfluoroalkanoic acid therein in a dry state, then,feeding a basic, nitrogen-containing aromatic cyclic compound or atertiary amine compound and water molecule both of vapor phase suppliedby using an aqueous solution in which the basic, nitrogen-containingaromatic cyclic compound or a tertiary amine compound is dissolved,allowing the water molecule to act on the reaction products of peptidein the presence of said basic, nitrogen-containing organic compound, andafter said treatment for hydrolysis re-drying post-treatment which isconducted by removing the basic, nitrogen-containing organic compoundand water molecule both remaining in the mixture containing a series ofthe reaction products, followed by drying.
 13. A method claimed in claim1 or claim 12, wherein, in addition to the step of said treatment usingthe mixture obtained by adding a small amount of the perfluoroalkanoicacid to the alkanoic acid anhydride, the method is provided, prior tothe step of releasing the C-terminal amino acids successively, with anadditional step for a pre-treatment of applying, to the N-terminal aminogroup of the peptide to be examined, N-acylation protection in advancewith use of an acyl group derived from the alkanoic acid constitutingsaid alkanoic acid anhydride.
 14. A method claimed in claim 13, whereinthe pretreatment step of applying N-acylation protection to theN-terminal amino group is conducted by employing technique where theN-acylation for amino groups in the peptide is effected by allowing analkanoic acid anhydride and an alkanoic acid both of vapor phase, whichare supplied from a mixture obtained by adding a small amount of thealkanoic acid to the alkanoic acid anhydride, to act on a dried sampleof the peptide to be examined in a dry atmosphere at a temperatureselected in a range of 10 to 60° C.
 15. A method claimed in claim 14,wherein as the alkanoic acid anhydride used in the pretreatment step ofapplying N-acylation protection to the N-terminal and as the alkanoicacid anhydride used in the step conducted thereafter of releasing theC-terminal amino acids successively, used is the same alkanoic acidanhydride.
 16. A process for preparation of a mixture containing aseries of reaction products obtainable by releasing the C-terminal aminoacids successively from a target peptide with use of chemical means,wherein the process is conducted to prepare the mixture containing aseries of reaction products obtainable by releasing the C-terminal aminoacids successively from the peptide by the chemical means comprisingsteps of: allowing an alkanoic acid anhydride and a perfluoroalkanoicacid both of vapor phase, which are supplied from a mixture of thealkanoic acid anhydride with a small amount of the perfluoroalkanoicacid added thereto, to act on a dried sample of the target peptide in adry atmosphere at a temperature selected in a range of 15 to 60° C., andcarrying out the release of the C-terminal amino acid in associationwith the process that at the C-terminal of the peptide, the formation ofa 5-oxazolone structure represented by the following general formula(III):

wherein R1 is a side chain of the C-terminal amino acid of the peptide,and R2 is a side chain of the amino acid residue positioned just beforesaid C-terminal amino acid, is followed by the cleavage of the5-oxazolone ring.
 17. A kit used for treatment of reaction to releasethe C-terminal amino acids successively from a target peptide bychemical means, wherein the kit for treatment of releasing theC-terminal amino acids successively is the kit being set up with acombination of: as a liquid reagent for the reaction of releasing theC-terminal amino acids successively, at least a mixture obtained by asmall amount of a perfluoroalkanoic acid to an alkanoic acid anhydride,or separately the alkanoic acid anhydride and the perfluoroalkanoic acidin combination for preparation of the mixture, a sample container forholding a sample of the target peptide to be treated therein, and areactor vessel which is provided with a liquid reagent-holding systemcapable of reserving said liquid reagent therein and capable ofmaintaining such a state that said liquid reagent makes no directcontact with said peptide sample held in the sample container and whichhas capacity to accommodate said sample container inside.